The objective of this project is to gain a better understanding of the surface presentation and mechanism of secretion of adenylate cyclase toxin (ACT), an essential virulence determinant of Bordetella pertussis. In contrast to other repeats-in-toxin (RTX) family members that are secreted to the extracellular milieu, ACT remains associated with the outer membrane of B. pertussis. Years of investigation of ACT in this laboratory and others have lead to novel discoveries regarding the structure and mechanism of action of this fascinating toxin. In order to understand completely the involvement of ACT in the virulence of B. pertussis, however, it will be necessary to study the activities of ACT in the context of B. pertussis organisms. The first Specific Aim of this proposal is to determine the orientation of ACT on the surface of B. pertussis and the role of specific domains of ACT on the ability of whole bacteria to intoxicate target cells. Specific Aim II is to examine the mechanism by which ACT is secreted, yet retained on the surface of B. pertussis. A variety of cellular and molecular biological approaches will be employed to accomplish these Aims. Information obtained from the experiments described in the Specific Aims proposed herein will begin to bridge the gap between our present knowledge of how soluble ACT exerts its biological effects on target cells and the mechanism of action of ACT associated with live B. pertussis, the location from which it acts during infection.